Dendritic cells (hereinafter referred to as “DC”) are the only antigen-presenting cells, or APC, able to induce primary immune responses (Cella, et al. 1997. “Origin, maturation and antigen presenting function of dendritic cells,” Curr Opin Immunol 9:10; Banchereau, J., and R. M. Steinman. 1998. “Dendritic cells and the control of immunity,” Nature 392:245). Circulating DC precursors circulate to tissues where they reside as immature, antigen-capturing cells with high endocytic and phagocytic activity. Following antigen uptake, DC migrate to the secondary lymphoid organs where they mature and become antigen-presenting cells able to select and activate naïve antigen-specific CD4+ T cells. This permits diversification of the response and activation of antigen-specific effectors such as antigen-specific cytotoxic T lymphocytes (hereinafter referred to as “CTL”) and B cells as well as non-specific effectors such as NK cells, macrophages and eosinophils (Sogn, J. A. 1998. “Tumor immunology: the glass is half full,” Immunity 9:757).
DC have been used to elicit an immune response against tumors. The induction of tumor immunity can be viewed as a three-step process that includes: 1) presentation of tumor associated antigens; 2) selection and activation of tumor associated antigens—specific T cells as well as non-specific effectors; 3) localizing tumor associated antigens—specific T cells to the tumor site; and 4) recognition of restriction elements leading to the elimination of tumor cells. U.S. Pat. No. 5,637,483 issued to Dranoff et al. discloses a method by which modified tumor cells expressing cytokines such as GM-CSF and IL-2 are irradiated or rendered proliferation incompetent and then administered to a patient to act as a stimulator or suppressor of a patient's systemic immune system. Experiments in mice have demonstrated the development of both protective and therapeutic anti-tumor responses induced by DC loaded with tumor associated antigens (Bell, et al. 1999. “Dendritic cells,” Adv Immunol 72:255). Furthermore, pilot clinical trials in humans show the feasibility of DC administration and the ability of peptide-loaded DC to induce peptide-specific T cell responses in patients with lymphoma, malignant melanoma and prostate carcinoma (Mukherji, et al. 1995. “Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells,” Proc Natl Acad Sci USA 92:8078; Hsu, et al. 1996. “Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells,” Nat Med 2:52; Gilboa, E., et al. 1998. “Immunotherapy of cancer with dendritic-cell-based vaccines,” Cancer Immunol Immunother 46:82; Nestle, et al. 1998. “Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells,” Nat Med 4:328; Tjoa, et al. 1998. “Evaluation of phase I/II clinical trials in prostate cancer with dendritic cells and PSMA peptides,” Prostate 36:39).
In order for DC to affect the immune system, the cells must possess processed peptide fragments to interact with T-cells, or be “loaded.” One of the critical challenges for the use of DC as immunotherapy vectors is the identification of an efficient antigen loading strategy. Several systems have been employed to deliver tumor associated antigens to DC including: 1) defined peptides of known sequences (Mayordomo, et al. 1995. “Bone marrow-derived dendritic cells pulsed with synthetic tumor peptides elicit protective and therapeutic antitumor immunity,” Nat Med 1:1297; Celluzzi, et al. 1996. “Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity,” J Exp Med 183:283); 2) undefined acid-eluted peptides from autologous tumor (Zitvogel, et al. 1996. “Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines,” J Exp Med 183:87); 3) whole tumor lysates (Fields, et al. 1998. “Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo,” Proc Natl Acad Sci USA 95:9482); 4) retroviral and adenoviral vectors (Song, et al. 1997. “Dendritic cells genetically modified with an adenovirus vector encoding the cDNA for a model antigen induce protective and therapeutic antitumor immunity,” J Exp Med 186:1247; Specht, et al. 1997. “Dendritic cells retrovirally transduced with a model antigen gene are therapeutically effective against established pulmonary metastases,” J Exp Med 186:1213); 5) tumor cell derived RNA (Boczkowski, et al. 1996. “Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo,” J Exp Med 184:465); and 6) fusion of DC with tumor cells (Gong, et al. 1998. “Reversal of tolerance to human MUCI antigen in MUCI transgenic mice immunized with fusions of dendritic and carcinoma cells, Proc Natl Acad Sci USA 95:6279).
Although all methods for delivering tumor associated antigens described above induce T cell responses and cause considerable anti-tumor effects, each has potential drawbacks (Sogn, J. A. 1998. “Tumor immunology: the glass is half full,” Immunity 9:757). Foremost, peptide-based strategies are limited by 1) the knowledge of the MHC haplotype of each patient; 2) the knowledge of the corresponding MHC class I binding motifs of the tumor associated antigens; 3) the variability of the MHC class I binding affinity of the synthetic peptides; and 4) the knowledge of whether or which various defined peptides represent tumor associated antigens in vivo.
Unlike a peptide-based approach which requires information about MHC haplotype and peptide sequence, unfractionated tumor tissue can provide both MHC class I and MHC class II epitopes without identifying tumor associated antigens. Recent studies demonstrated the ability of DC to capture dead or dying cells and elicit MHC class I-restricted secondary CTL responses (Albert, et al. 1998. “Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs,” Nature 392:86; Albert, et al. 1998. “Tumor-specific killer cells in paraneoplastic cerebellar degeneration,” Nat Med 4:1321). These studies indicated that 1) DC loaded with dead or dying influenza virus-infected cells can activate lymphocytes to mount virus-specific CTL responses; and 2) DC that capture dead or dying tumor cells can present antigen to tumor associated antigens peptide-specific T cells that were derived from patients with the paraneoplastic cerebellar degeneration. Because of the potential of engineered DC to contribute positively in the treatment of human disease, a simple method is desired for their production.
U.S. Pat. No. 5,851,756 issued to Steinman et al. discloses methods by which proliferating cultures of DC precursors and mature DC can be produced in vitro. Steinman et al. disclose the production of T cell dependent antigens using mature DC cultures, wherein the antigens are comprised of DC modified antigens or antigen-activated DC. Steinman et al. further disclose that the DC modified antigens or antigen-activated DC can be used as immunogens for vaccines or treatment of infectious diseases.
Major problems in tumor immunotherapy as experienced in the methods presented above are the limited number of well-defined tumor associated antigens and the lack of evidence that the known tumor associated antigens actually represent rejection antigens in vivo. Furthermore, the use of MHC class I binding peptides is associated with the HLA restriction and the limitation of induced immune responses to CD8+ T cells. In this context, the use of unfractionated antigenic material, in the form of dead or dying allogeneic tumor cells, that provides both MHC class I and class II epitopes leading to a diversified immune response involving many clones of CD4+ T cells and CTL represents an attractive alternative.
International Application No. WO 99/42564 of Albert et al. discloses methods directed toward developing therapies for increasing patient immunity to chronic infections and tumors by inducing tumor or infected cells to undergo apoptosis, having the apoptotic tumor or infected cells gain access to phagocytic, maturing dendritic cells, and exposing the apoptotic cell-primed dendritic cells expressing antigen of interest to T cells in vivo or in vitro for the induction of antigen-specific T cell responses.
We have now developed a method for engineering immunogenic DC. We have perfected a process using immature human antigen-presenting cells, including DC, derived from healthy volunteers and from patients that can capture dead or dying allogeneic cells, and subsequently present their antigens to autologous T cells, thus inducing proliferation of autologous CD4+ T cells and CD8+ t cells. Furthermore, by using the processes described herein, it has been found that DC loaded with allogeneic tumor cells can activate CD8+ T cells, generate CTL specific for antigens expressed by the dead or dying cells, and also recognize antigens that are shared between different tumor cell lines. Yet another finding is that by using the process described herein, DC loaded with dead or dying allogeneic tumor cell lines can prime naïve T cells and induce their differentiation to antigen-specific CTL.